Anti-il-6/il-6r antibodies and methods of use thereof

ABSTRACT

This invention provides fully human monoclonal antibodies that recognize the IL-6/IL-6R complex. The invention further provides methods of using such monoclonal antibodies as a therapeutic, diagnostic, and prophylactic.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/823,086, filed Nov. 27, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/993,153, filed Jan. 12, 2016, which is acontinuation of U.S. patent application Ser. No. 13/723,694, filed Dec.21, 2012 and issued as U.S. Pat. No. 9,234,034, which is a continuationof U.S. patent application Ser. No. 13/227,157, filed Sep. 7, 2011 andissued as U.S. Pat. No. 8,337,849, which is a continuation of U.S.patent application Ser. No. 12/465,295, filed May 13, 2009 and issued asU.S. Pat. No. 8,034,344, which claims the benefit of U.S. ProvisionalApplication No. 61/127,403, filed May 13, 2008, and U.S. ProvisionalApplication No. 61/194,156, filed Sep. 25, 2008, the contents of each ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to the generation of monoclonalantibodies, e.g., fully human monoclonal antibodies, that recognize theIL-6/IL-6R complex, to monoclonal antibodies, e.g., fully humanantibodies that recognize both the IL-6/IL-6R complex and IL-6R, and tomethods of using the monoclonal antibodies as therapeutics.

INCORPORATION BY REFERENCE

The contents of the text file named “TIZI-016D02US_SeqList.txt,” whichwas created on Jul. 29, 2020 and is 19.3 KB in size, are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Interleukin 6 (IL-6) is a potent pleiotropic cytokine that regulatescell growth and differentiation and is also an important mediator ofacute inflammatory responses. IL-6 exhibits its action via a receptorcomplex consisting of a specific IL-6 receptor (IL-6R) and a signaltransducing subunit (gp130). Dysregulated IL-6 signaling has beenimplicated in the pathogenesis of many diseases, such as multiplemyeloma, autoimmune diseases and prostate cancer. Accordingly, thereexists a need for therapies that neutralize the biological activities ofIL-6 and/or IL-6R.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies such as fully humanmonoclonal antibodies which recognize membrane bound human Interleukin-6(“IL-6”) when complexed with the human IL-6 receptor (i.e., the humanIL-6/IL-6R complex (“IL-6Rc”) (i.e., IL-6Rc expressed on the cellsurface or in soluble form). The antibodies of the invention are capableof modulating, e.g., blocking, inhibiting, reducing, antagonizing,neutralizing or otherwise interfering with IL-6R intracellular signalingvia activation of the JAK/STAT pathway and MAPK cascade. Antibodies ofthe invention also include antibodies that bind soluble IL-6Rc. Inaddition, antibodies of the invention include antibodies that bindIL-6Rc, wherein they also bind human IL-6R alone (i.e., when notcomplexed with IL-6).

The problem to be solved by the instant invention is the generation ofantibodies that bind the complex formed by IL-6R and IL-6 to therebyprevent the binding of the IL-6/IL-6R complex (“IL-6Rc”) to thetransmembrane glycoprotein gp130 and subsequent signaling (both cis andtrans), which is activated by the IL-6Rc/gp130 signaling complex.

The antibodies of the invention modulate, e.g., block, inhibit, reduce,antagonize, neutralize or otherwise interfere with, the interactionbetween the IL-6Rc and gp130. Binding of IL-6 and IL-6R to form theIL-6Rc complex allows the IL-6Rc to interact or otherwise associate withgp130, a transmembrane glycoprotein. In particular, binding of IL-6 toIL-6R leads to disulfide-linked homodimerization of gp130 within a cell,which, in turn, leads to the activation of a tyrosine kinase as thefirst step in signal transduction. In a preferred embodiment, theantibodies of the invention bind to IL-6Rc and block or otherwiseinhibit IL-6Rc from interacting with gp130, thereby preventing,partially or completely, the homodimerization of gp130 and subsequentsignaling (cis and trans).

Unlike antibodies that bind to IL-6 or IL-6R individually, for example,in the groove where IL-6 binds to IL-6R, the antibodies of the inventiondo not inhibit or otherwise interfere with the interaction between IL-6and IL-6R to form the IL-6Rc complex. The antibodies of the inventionare, therefore, used at concentrations that are significantly lower thanthe concentrations needed for antibodies that block or otherwiseinterfere with the interaction between IL-6R and IL-6, for example,antibodies that compete with IL-6 for binding to IL-6R or vice versa. Insome embodiments, the concentration of the antibodies of the inventionis 50-100 times lower than the concentration needed for an antibody thatblocks or otherwise interferes with the interaction between IL-6 andIL-6R. For antibodies that block or otherwise interfere with theinteraction between IL-6 and IL-6R, a large concentration must be used,for example, to treat inflammation, where the levels of IL-6R increaseand/or the expression of IL-6 increases. To compete effectively and overa prolonged period with the increased levels of IL-6 and/or IL-6R, theseantibodies that block or otherwise interfere with the interactionbetween IL-6 and IL-6R must be present in large concentrations.

Exemplary monoclonal antibodies of the invention include, for example,the 39B9 VL1 antibody, the 39B9 VL5 antibody, the 12A antibody and the5C antibody. Alternatively, the monoclonal antibody is an antibody thatbinds to the same epitope as the 39B9 VL1 antibody, the 39B9 VL5antibody, the 12A antibody and the 5C antibody. These antibodies arerespectively referred to herein as “huIL-6Rc” antibodies. huIL-6Rcantibodies include fully human monoclonal antibodies, as well ashumanized monoclonal antibodies and chimeric antibodies. Theseantibodies show specificity for human IL-6Rc and IL-6R, and they havebeen shown to modulate, e.g., block, inhibit, reduce, antagonize,neutralize or otherwise interfere with IL-6Rc mediated intracellularsignaling (cis and/or trans signaling).

In a preferred embodiment, the fully human antibodies of the inventioninclude (i) the consensus amino acid sequence QQSXSYPLT (SEQ ID NO: 42)in the light chain complementarity determining region 3 (CDR3), where Xis N or Q; (ii) the consensus amino acid sequence GIIPX₁FX₂TTKYAQX₃FQG(SEQ ID NO: 43) in the heavy chain complementarity determining region 2(CDR2), where X₁ is L or A, X2 is D or E, and X3 is Q or K; (iii) theconsensus amino acid sequence DRDILTDYYPXGGMDV (SEQ ID NO: 44) in theheavy chain complementarity determining region 3 (CDR3), where X is M orL; and (iv) the consensus amino acid sequence TAVXYCAR (SEQ ID NO: 45)in the framework region 3 (FRW3), where X is F or Y.

For example, in one of the preferred embodiments, the huIL-6Rc antibodyincludes the amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the lightchain CDR3 region, the amino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO:33) in the heavy chain CDR2 region, the amino acid sequenceDRDILTDYYPMGGMDV (SEQ ID NO: 36) in the heavy chain CDR3 region, and theamino acid sequence TAVYYCAR (SEQ ID NO: 39) in the FRW3 region. Thisantibody is referred to herein as the NI-1201A antibody.

In another of the preferred embodiments, the huIL-6Rc antibody includesthe amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chainCDR3 region, the amino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 33)in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV(SEQ ID NO: 37) in the heavy chain CDR3 region, and the amino acidsequence TAVYYCAR (SEQ ID NO: 39) in the FRW3 region. This antibody isreferred to herein as the NI-1201B antibody.

In another of the preferred embodiments, the huIL-6Rc antibody includesthe amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chainCDR3 region, the amino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 34)in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV(SEQ ID NO: 37) in the heavy chain CDR3 region, and the amino acidsequence TAVYYCAR (SEQ ID NO: 39) in the FRW3 region. This antibody isreferred to herein as the NI-1201C antibody.

In another of the preferred embodiments, the huIL-6Rc antibody includesthe amino acid sequence QQSQSYPLT (SEQ ID NO: 32) in the light chainCDR3 region, the amino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 34)in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPLGGMDV(SEQ ID NO: 37) in the heavy chain CDR3 region, and the amino acidsequence TAVYYCAR (SEQ ID NO: 39) in the FRW3 region. This antibody isreferred to herein as the NI-1201D antibody.

In other embodiments, the huIL-6Rc antibody includes the amino acidsequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, theamino acid sequence GIIPLFDTTKYAQQFQG (SEQ ID NO: 16) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPMGGMDV (SEQ ID NO: 36) inthe heavy chain CDR3 region, and the amino acid sequence TAVFYCAR (SEQID NO: 38) in the FRW3 region. This antibody is referred to herein asthe NI-1201 wild type (NI-1201-WT) antibody.

The fully human antibodies of the invention contain a heavy chainvariable region having the amino acid sequence of SEQ ID NOS: 2, 8, and12. The fully human antibodies of the invention contain a light chainvariable region having the amino acid sequence of SEQ ID NOS: 4, 6, 10and 14. The antibody binds to IL-6R, to IL-6R complexed with IL-6 (i.e.,IL-6Rc) or both.

The three heavy chain CDRs include a variable heavy chain (VH)complementarity determining region 1 (CDR1) that includes an amino acidsequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to asequence selected from the group consisting of SEQ ID NOs: 15, 18 and21; a VH complementarity determining region 2 (CDR2) that includes anamino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or moreidentical to a sequence selected from the group consisting of SEQ IDNOs: 16, 19, 22, 33, 34 and 35; and a VH complementarity determiningregion 3 (CDR3) that includes an amino acid sequence at least 90%, 92%,95%, 97% 98%, 99% or more identical to a sequence selected from thegroup consisting of SEQ ID NOs: 17, 20, 23, 36 and 37. The antibodybinds to IL-6R, to IL-6R complexed with IL-6 (i.e., IL-6Rc) or both.

The three light chain CDRs include variable light chain (VL) CDR1 thatincludes an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% ormore identical to a sequence selected from the group consisting of SEQID NOs: 24, 27, 28, and 30; a VL CDR2 that includes an amino acidsequence at least 90%, 92%, 95%, 97% 98%, 99% or more identical to theamino acid sequence of SEQ ID NO: 25; and a VL CDR3 that includes anamino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% or moreidentical to a sequence selected from the group consisting of SEQ IDNOs: 26, 29, 31 and 32. The antibody binds to IL-6R, to IL-6R complexedwith IL-6 (i.e., IL-6Rc) or both.

The huIL-6Rc antibodies provided herein are fully human antibodies thatbind to IL-6/IL-6R complex (IL-6Rc) and prevent IL-6Rc from binding togp130 such that gp130-mediated intracellular signaling cascade is notactivated in the presence of these antibodies. Preferably, theantibodies have an affinity of at least 1×10⁻⁸ for IL-6Rc, and morepreferably, the antibodies have an affinity of at least 1×10⁻⁹ forIL-6Rc.

Antibodies of the invention immunospecifically bind IL-6Rc wherein theantibody binds to an epitope that includes one or more amino acidresidues on human IL-6 and/or human IL-6R. Antibodies of the inventionimmunospecifically binds both IL-6Rc and IL-6R, wherein the antibodybinds to an epitope that includes one or more amino acid residues onhuman IL-6 and/or human IL-6R. Preferably, the huIL-6Rc antibodiesdescribed herein bind to an epitope in domain 3 of IL-6 receptor(IL-6R). More preferably, the epitope to which the huIL-6Rc antibodiesbind includes at least the amino acid sequence AERSKT (SEQ ID NO: 46).

Antibodies of the invention also include fully human antibodies thatspecifically bind IL-6Rc, and antibodies that specifically bind bothIL-6Rc and IL-6R, wherein the antibody exhibits greater than 50%inhibition of IL-6 mediated activation of the JAK/STAT pathway and MAPKcascade. For example, antibodies of the invention exhibit greater than55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%inhibition of IL-6 mediated functions including STAT3 activation, acutephase protein production, antibody production and cellulardifferentiation and/or proliferation.

The present invention also provides methods of treating or preventingpathologies associated with aberrant IL-6 receptor activation and/oraberrant IL-6 signaling (cis and/or trans) or alleviating a symptomassociated with such pathologies, by administering a monoclonal antibodyof the invention (e.g., fully human monoclonal antibody) to a subject inwhich such treatment or prevention is desired. The subject to be treatedis, e.g., human. The monoclonal antibody is administered in an amountsufficient to treat, prevent or alleviate a symptom associated with thepathology. The amount of monoclonal antibody sufficient to treat orprevent the pathology in the subject is, for example, an amount that issufficient to reduce IL-6Rc induced activation of the JAK/STAT pathwayor MAPK cascade. For example, IL-6Rc induced activation of the JAK/STATpathway or MAPK cascade is decreased when the level of STAT3 activationin the presence of a monoclonal antibody of the invention is greaterthan or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,90%, 95%, 99%, or 100% lower than a control level of STAT3 activation(i.e., the level of STAT3 activation in the absence of the monoclonalantibody). Those skilled in the art will appreciate that the level ofSTAT3 activation can be measured using a variety of assays, including,for example, commercially available ELISA kits.

Pathologies treated and/or prevented using the monoclonal antibodies ofthe invention (e.g., fully human monoclonal antibody) include, forexample, sepsis, cancer (e.g., multiple myeloma disease (MM), renal cellcarcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferativedisorder (BLPD), and prostate cancer), bone resorption, osteoporosis,cachexia, psoriasis, mesangial proliferative glomerulonephritis,Kaposi's sarcoma, AIDS-related lymphoma, and inflammatory diseases(e.g., rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus).

Pharmaceutical compositions according to the invention can include anantibody of the invention and a carrier. These pharmaceuticalcompositions can be included in kits, such as, for example, diagnostickits.

One skilled in the art will appreciate that the antibodies of theinvention have a variety of uses. For example, the proteins of theinvention are used as therapeutic agents to prevent IL-6 receptoractivation in disorders such as, for example, sepsis, cancer (e.g.,multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma cellleukaemia, lymphoma, B-lymphoproliferative disorder (BLPD), and prostatecancer), bone resorption, osteoporosis, cachexia, psoriasis, mesangialproliferative glomerulonephritis, Kaposi's sarcoma, AIDS-relatedlymphoma, and inflammatory diseases (e.g., rheumatoid arthritis,systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia,Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE),multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma,asthma, allergic asthma and autoimmune insulin-dependent diabetesmellitus). The antibodies of the invention are also used as reagents indiagnostic kits or as diagnostic tools, or these antibodies can be usedin competition assays to generate therapeutic reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are a series of graphs depicting the ability of an antibodyof the invention, NI-1201, to block IL-6 trans-signaling.

FIGS. 2A and 2B are a series of graphs depicting the ability of theNI-1201 antibody to block IL-6 cis-signaling.

FIGS. 3A and 3B are a series of illustrations depicting the ability ofthe NI-1201 antibody to block STAT-3 phosphorylation induced by IL-6cis-signaling.

FIG. 4 is a graph depicting the ability of the NI-1201 antibody to blockIL-6 trans-signaling mediated by the fusion protein of soluble humanIL-6/IL-6R complex (“shuIL-6Rc”).

FIG. 5 is a graph depicting the binding of the NI-1201 antibody tomembrane-bound IL-6R.

FIGS. 6A-6D are a series of illustrations and graphs depicting themapping of the NI-1201 epitope on IL-6R.

FIGS. 7A and 7B are an illustration and a graph depicting the ability ofthe NI-1201 antibody to cross-react and neutralize cynomolgus monkeyIL-6 signaling.

DETAILED DESCRIPTION

The present invention provides monoclonal antibodies that specificallybind the human IL-6/IL-6 receptor complex (“IL-6Rc”), in soluble form,or membrane bound (i.e., when expressed on a cell surface). Theinvention further provides monoclonal antibodies that specifically bindIL-6Rc, wherein the antibodies also bind IL-6R when not complexed withIL-6. These antibodies are collectively referred to herein as “huIL-6Rc”antibodies. The antibody is e.g., a fully human antibody.

Antibodies of the invention specifically bind IL-6Rc and/or both IL-6Rcand IL-6R wherein the antibody binds to an epitope that includes one ormore amino acid residues of human IL-6, IL-6R, or both.

The antibodies of the present invention bind to an IL-6Rc and/or bothIL-6Rc and IL-6R epitope with an equilibrium binding constant (K_(d)) of≤1 μM, e.g., ≤100 nM, preferably ≤10 nM, and more preferably ≤1 nM. Forexample, the huIL-6Rc antibodies provided herein exhibit a K_(d) in therange approximately between ≤1 nM to about 1 pM.

IL-6 acts as both a pro-inflammatory and anti-inflammatory cytokine. Itis secreted by T cells and macrophages to stimulate immune response totrauma, especially burns or other tissue damage leading to inflammation.IL-6 is one of the most important mediators of fever and of the acutephase response. In the muscle and fatty tissue IL-6 stimulates energymobilization which leads to increased body temperature. IL-6 can besecreted by macrophages in response to specific microbial molecules,referred to as pathogen associated molecular patterns (PAMPs). ThesePAMPs bind to highly important detection molecules of the innate immunesystem, called Toll-like receptors (TLRs), that are present on the cellsurface (or in intracellular compartments) which induce intracellularsignaling cascades that give rise to inflammatory cytokine production.IL-6 is also essential for hybridoma growth and is found in manysupplemental cloning media such as briclone.

IL-6 signals through a cell-surface type I cytokine receptor complexconsisting of the ligand-binding IL-6Rα chain (also called known asCD126), and the signal-transducing component gp130 (also called CD130).gp130 is the common signal transducer for several cytokines includingleukemia inhibitory factor (LIF), ciliary neurotrophic factor,oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitouslyexpressed in most tissues. In contrast, the expression of CD126 isrestricted to certain tissues. As IL-6 interacts with its receptor, ittriggers the gp130 and IL-6R proteins to form a complex, thus activatingthe receptor. These complexes bring together the intracellular regionsof gp130 to initiate a signal transduction cascade through certaintranscription factors, Janus kinases (JAKs) and Signal Transducers andActivators of Transcription (STATs). Accordingly, neutralization of IL-6signaling is a potential therapeutic strategy in the treatment ofdisorders such as, for example, sepsis, cancer (e.g., multiple myelomadisease (MM), renal cell carcinoma (RCC), plasma cell leukaemia,lymphoma, B-lymphoproliferative disorder (BLPD), and prostate cancer),bone resorption, osteoporosis, cachexia, psoriasis, mesangialproliferative glomerulonephritis, Kaposi's sarcoma, AIDS-relatedlymphoma, and inflammatory diseases (e.g., rheumatoid arthritis,systemic onset juvenile idiopathic arthritis, hypergammaglobulinemia,Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE),multiple sclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma,asthma, allergic asthma and autoimmune insulin-dependent diabetesmellitus).

The huIL-6Rc antibodies of the invention serve to modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere with thefunctional activity of IL-6Rc. Functional activities of IL-6Rc includefor example, intracellular signaling via activation of the JAK/STATpathway and activation of the MAPK cascade, acute phase proteinproduction, antibody production and cellular differentiation and/orproliferation. For example, the huIL-6Rc antibodies completely orpartially inhibit IL-6Rc functional activity by partially or completelymodulating, blocking, inhibiting, reducing antagonizing, neutralizing,or otherwise interfering with the binding of IL-6Rc to thesignal-transducing receptor component gp130.

The huIL-6Rc antibodies are considered to completely modulate, block,inhibit, reduce, antagonize, neutralize or otherwise interfere withIL-6Rc functional activity when the level of IL-6Rc functional activityin the presence of the huIL-6Rc antibody is decreased by at least 95%,e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of IL-6Rcfunctional activity in the absence of binding with a huIL-6Rc antibodydescribed herein. The huIL-6Rc antibodies are considered to partiallymodulate, block, inhibit, reduce, antagonize, neutralize or otherwiseinterfere with IL-6Rc functional activity when the level of IL-6Rcactivity in the presence of the huIL-6Rc antibody is decreased by lessthan 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%as compared to the level of IL-6Rc activity in the absence of bindingwith a huIL-6Rc antibody described herein.

Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the terms Interleukin-6 Receptor, IL-6R, Interleukin-6Receptor-alpha, IL-6Rα, cluster differentiation factor 126, and CD126are synonymous and may be used interchangeably. Each of these termsrefers to the homodimeric protein, except where otherwise indicated.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” “or directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at much loweraffinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or fragment thereof, ora T-cell receptor. The term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. An antibody is said tospecifically bind an antigen when the dissociation constant is ≤1 μM;e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to IL-6Rc and/or both IL-6Rc and IL-6R, whenthe equilibrium binding constant (K_(d)) is ≤1 μM, preferably ≤100 nM,more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM, asmeasured by assays such as radioligand binding assays or similar assaysknown to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules presented in SEQ IDNOS: 2, 8 and 12, and nucleic acid molecules encoding the light chainimmunoglobulin molecules represented in SEQ ID NOS: 4, 6, 10, and 14.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules represented in SEQ ID NOS: 2, 8, and 12,and the light chain immunoglobulin molecules represented in SEQ ID NOS:4, 6, 10, and 14 as well as antibody molecules formed by combinationscomprising the heavy chain immunoglobulin molecules with light chainimmunoglobulin molecules, such as kappa light chain immunoglobulinmolecules, and vice versa, as well as fragments and analogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has specific bindingto IL-6Rc and/or both IL-6Rc and IL-6R, under suitable bindingconditions. Typically, polypeptide analogs comprise a conservative aminoacid substitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:— CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—,CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used to generate more stable peptides. In addition, constrainedpeptides comprising a consensus sequence or a substantially identicalconsensus sequence variation may be generated by methods known in theart (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I) orradionuclides (e.g., fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Ménière's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, chronic obstructive pulmonary disease,atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema,glomerulonephritis, graft vs. host disease, hemolytic anemias,osteoarthritis, sepsis, stroke, transplantation of tissue and organs,vasculitis, diabetic retinopathy and ventilator induced lung injury.

Cancers include, for example, multiple myeloma disease (MM), renal cellcarcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferativedisorder (BLPD), and prostate cancer.

huIL-6Rc Antibodies

Monoclonal antibodies of the invention (e.g., fully human monoclonalantibodies) have the ability to inhibit IL-6Rc mediated cell signaling.Inhibition is determined, for example, using the cellular assaydescribed herein in Example 1 and 2.

Exemplary antibodies of the invention include, for example, the 39B9 VL1antibody, the 39B9 VL5 antibody, the 12A antibody, and the 5C antibody.These antibodies show specificity for human IL-6Rc and/or both IL-6Rcand IL-6R and they have been shown to inhibit the functional activity ofIL-6Rc (i.e., binding to gp130 to induce the signaling cascade) invitro.

Each of the huIL-6Rc monoclonal antibodies described herein includes aheavy chain variable region (VH) and a light chain variable region (VL),as shown in the amino acid and corresponding nucleic acid sequenceslisted below.

The 39B9 VL1 and 39B9 VL5 antibodies share a common heavy chain variableregion (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQID NO:1

>39B9 VL1-VH nucleic acid sequence (SEQ ID NO: 1)5′CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGCCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTCTCTTTGATACAACAAAGTACGCACAGCAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTATTTTACTGTGCGAGAGATCGGGATATTTTGACTGATTATTATCCCATGGGCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 3′ >39B9 VL1-VH amino acid sequence(SEQ ID NO: 2) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPLFDTTKYAQQFQGRVTITADESTSTAYMELSSLRSEDTAVFYCARDRDILTDYYPMGGMDVWGQGTTVTVSS

The 39B9 VL1 antibody includes a light chain variable region (SEQ IDNO:4) encoded by the nucleic acid sequence shown in SEQ ID NO:3.

>39B9 VL1-VL nucleic acid sequence (SEQ ID NO: 3)5′GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGTTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGT 3′ >39B9 VL1-VL amino acid sequence(SEQ ID NO: 4) AIQLTQSPSSLSASVGDRVTITCRASQGISSVLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSYPLTFGG GTKVEIKR

The 39B9 VL5 antibody includes a light chain variable region (SEQ IDNO:6) encoded by the nucleic acid sequence shown in SEQ ID NO:5.

>39B9 VL5-VL nucleic acid sequence (SEQ ID NO: 5)5′GACATCCTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTAATAGTTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA 3′ >39B9 VL5-VL amino acid sequence(SEQ ID NO: 6) DILMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNSYPLTFGG GTKVEIKR

The 12A antibody includes a heavy chain variable region (SEQ ID NO:8)encoded by the nucleic acid sequence shown in SEQ ID NO:7.

>12A VH nucleic acid sequence (SEQ ID NO: 7)5′CAGGTGCAGCTGGTGGAGTCTTGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGACATGTACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATTAGATGATGGAAATAATAATTACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAAAAAGGTGTATCTGCAAATGAATAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAGAGCGTCCCCTAACTGGGGTCTTCTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 3′ >12A VH amino acid sequence (SEQ ID NO: 8)QVQLVESWGGVVQPGRSLRLSCAASGFTFSNYDMYWVRQAPGKGLEWVAVILDDGNNNYYADSVKGRFTISRDNSKKKVYLQMNSLRAEDTAVYYCVRAS PNWGLLDFWGQGTLVTVSS

The 12A antibody includes a light chain variable region (SEQ ID NO:10)encoded by the nucleic acid sequence shown in SEQ ID NO:9.

>12A VL nucleic acid sequence (SEQ ID NO: 9)5′GAAATTGTGTTGACACAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGT 3′ >12A VL amino acid sequence(SEQ ID NO: 10) EIVLTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPITFGQ GTRLEIKR

The 5C antibody includes a heavy chain variable region (SEQ ID NO:12)encoded by the nucleic acid sequence shown in SEQ ID NO:11

>5C VH nucleic acid sequence (SEQ ID NO: 11)CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATCTTCAGTAGCTATGACATGTACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATTATATGATGGAAATAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGTGAGAGCGTCCCCTAACTGGGGTCTTTTTGACTTCTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGT 3′ >5C VH amino acid sequence (SEQ ID NO: 12)QVQLVQSGGGVVQPGRSLRLSCAASGFIFSSYDMYWVRQAPGKGLEWVAVILYDGNNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCVRAS PNWGLFDFWGQGTLVTVSS

The 5C antibody includes a light chain variable region (SEQ ID NO:14)encoded by the nucleic acid sequence shown in SEQ ID NO:13.

>5C VL nucleic acid sequence (SEQ ID NO: 13)5′GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATGTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGT 3′ >5C VL amino acid sequence (SEQ ID NO: 14)DIQMTQSPSSLSASVGDRVTITCRASQGISSDLAWYQQKPGKAPKLLMYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPITFGQ GTRLEIKR

huIL-6Rc antibodies of the invention additionally comprise, for example,the heavy chain complementarity determining regions (VH CDRs) shownbelow in Table 1, the light chain complementarity determining regions(VL CDRs) shown in Table 2, and combinations thereof.

TABLE 1 VH CDR sequences from antibody clones that bind and neutralizeIL-6Rc biological activity Clone Name VH CDR1 VH CDR2 VH CDR3 39B9 SYAISGIIPLFDTTKYAQQFQG CAR DRDILTDYYPMGGMDV (SEQ ID NO: 15) (SEQ ID NO: 16)(SEQ ID NO: 17) 12A NYDMY VILDDGNNNYYADSVKG CVR ASPNWGLLDF(SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) 5C SYDMYVILYDGNNKYYADSVKG CVR ASPNWGLFDF (SEQ ID NO: 21) (SEQ ID NO: 22)(SEQ ID NO: 23)

TABLE 2 VL CDR sequences from antibody clones that bind and neutralizeIL-6Rc Clone Name VL CDR1 VL CDR2 VL CDR3 39B9 VL1 RASQGISSVLA DASSLESQQSNSYPLT (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) 39B9 VL5RASQDISSWLA DASSLES QQSNSYPLT (SEQ ID NO: 27) (SEQ ID NO: 25)(SEQ ID NO: 26) 12A RASQGISSWLA DASSLES QQSNSYPIT (SEQ ID NO: 28)(SEQ ID NO: 25) (SEQ ID NO: 29) 5C RASQGISSVDA DASSLES QQSNSYPIT(SEQ ID NO: 30) (SEQ ID NO: 25) (SEQ ID NO: 31)

The amino acids encompassing the complementarity determining regions(CDR) are as defined by E. A. Kabat et al. (See Kabat, E A, et al.,Sequences of Protein of immunological interest, Fifth Edition, USDepartment of Health and Human Services, US Government Printing Office(1991)).

Also included in the invention are antibodies that bind to the sameepitope as the antibodies described herein. For example, antibodies ofthe invention specifically bind to IL-6R, wherein the antibody binds toan epitope that includes one or more amino acid residues on human IL-6R(e.g., GenBank Accession No. P08887). Antibodies of the inventionspecifically bind IL-6Rc, wherein the antibody binds to an epitope thatincludes one or more amino acid residues on human IL-6 (e.g., GenBankAccession No. NP_000591), IL-6R (e.g., GenBank Accession No. P08887), orboth.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a monoclonal antibody(e.g., fully human monoclonal antibody) has the same specificity as amonoclonal antibody of the invention (e.g., clones 39B9 VL1, 39B9 VL5,12A and 5C) by ascertaining whether the former prevents the latter frombinding to gp130. If the monoclonal antibody being tested competes withthe monoclonal antibody of the invention, as shown by a decrease inbinding by the monoclonal antibody of the invention, then the twomonoclonal antibodies bind to the same, or a closely related, epitope.

An alternative method for determining whether a monoclonal antibody hasthe specificity of monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with solubleIL-6Rc or IL-6R protein (with which it is normally reactive), and thenadd the monoclonal antibody being tested to determine if the monoclonalantibody being tested is inhibited in its ability to bind IL-6Rc and/orboth IL-6Rc and IL-6R. If the monoclonal antibody being tested isinhibited then, in all likelihood, it has the same, or functionallyequivalent, epitopic specificity as the monoclonal antibody of theinvention.

Screening of monoclonal antibodies of the invention, can be also carriedout, e.g., by measuring IL-6 receptor mediated activation of theJAK/STAT pathways and/or MAPK signaling cascade, and determining whetherthe test monoclonal antibody is able to modulate, block, inhibit,reduce, antagonize, neutralize or otherwise interfere with IL-6signaling.

Various procedures known within the art may be used for the productionof monoclonal antibodies directed against IL-6Rc and/or both IL-6Rc andIL-6R, or against derivatives, fragments, analogs homologs or orthologsthereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E,and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference). Fully human antibodiesare antibody molecules in which the entire sequence of both the lightchain and the heavy chain, including the CDRs, arise from human genes.Such antibodies are termed “human antibodies”, or “fully humanantibodies” herein. Human monoclonal antibodies are prepared, forexample, using the procedures described in the Examples provided below.Human monoclonal antibodies can be also prepared by using the triomatechnique; the human B-cell hybridoma technique (see Kozbor, et al.,1983 Immunol Today 4: 72); and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Humanmonoclonal antibodies may be utilized and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The antibodies of the invention (e.g., 39B9 VL1, 39B9 VL5, 12A and 5C)are fully human monoclonal antibodies. Monoclonal antibodies thatmodulate, block, inhibit, reduce, antagonize, neutralize or otherwiseinterfere with IL-6Rc mediated cell signaling are generated, e.g., byimmunizing an animal with membrane bound and/or soluble IL-6Rc, such as,for example, murine, rat or human IL-6Rc or an immunogenic fragment,derivative or variant thereof. Alternatively, the animal is immunizedwith cells transfected with a vector containing a nucleic acid moleculeencoding IL-6Rc such that IL-6Rc is expressed and associated with thesurface of the transfected cells. Alternatively, the antibodies areobtained by screening a library that contains antibody or antigenbinding domain sequences for binding to IL-6Rc. This library isprepared, e.g., in bacteriophage as protein or peptide fusions to abacteriophage coat protein that is expressed on the surface of assembledphage particles and the encoding DNA sequences contained within thephage particles (i.e., “phage displayed library”). Hybridomas resultingfrom myeloma/B cell fusions are then screened for reactivity to IL-6Rcand/or both IL-6Rc and IL-6R.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of monoclonalantibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (MA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Human Antibodies and Humanization of Antibodies

Monoclonal antibodies of the invention include fully human antibodies orhumanized antibodies. These antibodies are suitable for administrationto humans without engendering an immune response by the human againstthe administered immunoglobulin.

A huIL-6Rc antibody is generated, for example, using the proceduresdescribed in the Examples provided below.

In other, alternative methods, a huIL-6Rc antibody is developed, forexample, using phage-display methods using antibodies containing onlyhuman sequences. Such approaches are well-known in the art, e.g., inWO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporated byreference. In this approach, a combinatorial library of phage carryingrandom pairs of light and heavy chains are screened using natural orrecombinant source of IL-6Rc or fragments thereof. In another approach,a huIL-6Rc antibody can be produced by a process wherein at least onestep of the process includes immunizing a transgenic, non-human animalwith human IL-6Rc protein. In this approach, some of the endogenousheavy and/or kappa light chain loci of this xenogenic non-human animalhave been disabled and are incapable of the rearrangement required togenerate genes encoding immunoglobulins in response to an antigen. Inaddition, at least one human heavy chain locus and at least one humanlight chain locus have been stably transfected into the animal. Thus, inresponse to an administered antigen, the human loci rearrange to providegenes encoding human variable regions immunospecific for the antigen.Upon immunization, therefore, the xenomouse produces B-cells thatsecrete fully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. Nos. 6,075,181and 6,150,584, which is hereby incorporated by reference in itsentirety. This general strategy was demonstrated in connection withgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994), which is herebyincorporated by reference in its entirety. See also, U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and EuropeanPatent No., EP 0 463 151 B1 and International Patent Applications No. WO94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related familymembers.

In an alternative approach, others have utilized a “minilocus” approachin which an exogenous Ig locus is mimicked through the inclusion ofpieces (individual genes) from the Ig locus. Thus, one or more VH genes,one or more DH genes, one or more JH genes, a mu constant region, and asecond constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. See e.g., U.S. Pat. Nos.5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825; 5,625,126;5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215;5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458;and European Patent No. 0 546 073 B1; and International PatentApplication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO98/24884 and related family members.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a immune variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against IL-6Rc and/or both IL-6Rc and IL-6R in order tovitiate or otherwise mitigate concerns and/or effects of HAMA or HACAresponse.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization, chimerization and display techniquesusing appropriate libraries. It will be appreciated that murineantibodies or antibodies from other species can be humanized orprimatized using techniques well known in the art. See e.g., Winter andHarris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews inImmunol. 12125-168 (1992). The antibody of interest may be engineered byrecombinant DNA techniques to substitute the CH1, CH2, CH3, hingedomains, and/or the framework domain with the corresponding humansequence (See WO 92102190 and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,761; 5,693,792; 5,714,350; and 5,777,085). Also, the use of IgcDNA for construction of chimeric immunoglobulin genes is known in theart (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521(1987)). mRNA is isolated from a hybridoma or other cell producing theantibody and used to produce cDNA. The cDNA of interest may be amplifiedby the polymerase chain reaction using specific primers (U.S. Pat. Nos.4,683,195 and 4,683,202). Alternatively, a library is made and screenedto isolate the sequence of interest. The DNA sequence encoding thevariable region of the antibody is then fused to human constant regionsequences. The sequences of human constant regions genes may be found inKabat et al. (1991) Sequences of Proteins of immunological Interest,N.I.H. publication no. 91-3242. Human C region genes are readilyavailable from known clones. The choice of isotype will be guided by thedesired effecter functions, such as complement fixation, or activity inantibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1,IgG3 and IgG4. Either of the human light chain constant regions, kappaor lambda, may be used. The chimeric, humanized antibody is thenexpressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g., SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as willbe appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes andPlückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley andSmith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol.17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel etal. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A(1992), and U.S. Pat. No. 5,733,743. If display technologies areutilized to produce antibodies that are not human, such antibodies canbe humanized as described above.

Using these techniques, antibodies can be generated to IL-6Rc expressingcells, soluble forms of IL-6Rc, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed herein.

The huIL-6Rc antibodies of the invention can be expressed by a vectorcontaining a DNA segment encoding the single chain antibody describedabove.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofIL-6Rc and/or IL-6R in a sample. The antibody can also be used to try tobind to and disrupt IL-6Rc-related signaling.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F(ab′)2 fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F(ab′)2 fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2)anti-IL-6R fragments or anti-IL-6Rc complex fragments, single chainanti-IL-6R or anti-IL-6Rc antibodies, bispecific anti-IL-6R, and/oranti-IL-6Rc antibodies, and heteroconjugate anti-IL-6R and/oranti-IL-6Rc antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for IL-6Rc or IL-6R. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)2 fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberrantIL-6 signaling. For example, cysteine residue(s) can be introduced intothe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). (See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that hasdual Fc regions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NETS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. F_(ab′) fragments of the antibody of the present invention canbe conjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against IL-6Rc

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman WN “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

In one embodiment, antibodies of the invention, which include amonoclonal antibody of the invention (e.g., a fully human monoclonalantibody), may be used as therapeutic agents. Such agents will generallybe employed to diagnose, prognose, monitor, treat, alleviate, and/orprevent a disease or pathology associated with aberrant IL-6 signalingin a subject. A therapeutic regimen is carried out by identifying asubject, e.g., a human patient suffering from (or at risk of developing)a disease or disorder associated with aberrant IL-6 signaling, e.g., aninflammatory disorder such as rheumatoid arthritis, using standardmethods. An antibody preparation, preferably one having high specificityand high affinity for its target antigen, is administered to the subjectand will generally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withthe signaling function of the target (e.g., IL-6Rc). Administration ofthe antibody may abrogate or inhibit or interfere with the binding ofthe target (e.g., IL-6Rc) with an endogenous ligand (e.g., gp130) towhich it naturally binds. For example, the antibody binds to the targetand modulates, blocks, inhibits, reduces, antagonizes, neutralizes, orotherwise interferes with IL-6 signaling.

Diseases or disorders related to aberrant IL-6 signaling include sepsis,cancer (e.g., multiple myeloma disease (MM), renal cell carcinoma (RCC),plasma cell leukaemia, lymphoma, B-lymphoproliferative disorder (BLPD),and prostate cancer), bone resorption, osteoporosis, cachexia,psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma,AIDS-related lymphoma, and inflammatory diseases (e.g., rheumatoidarthritis, systemic onset juvenile idiopathic arthritis,hypergammaglobulinemia, Crohn's disease, ulcerative colitis, systemiclupus erythematosus (SLE), multiple sclerosis, Castleman's disease, IgMgammopathy, cardiac myxoma, asthma, allergic asthma and autoimmuneinsulin-dependent diabetes mellitus).

Symptoms associated with inflammatory-related disorders include, forexample, inflammation, fever, general malaise, fever, pain, oftenlocalized to the inflamed area, rapid pulse rate, joint pain or aches(arthralgia), rapid breathing or other abnormal breathing patterns,chills, confusion, disorientation, agitation, dizziness, cough, dyspnea,pulmonary infections, cardiac failure, respiratory failure, edema,weight gain, mucopurulent relapses, cachexia, wheezing, headache, andabdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation. Symptoms associated with immune-related disorders include,for example, inflammation, fever, loss of appetite, weight loss,abdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation, joint pain or aches (arthralgia), fatigue, rash, anemia,extreme sensitivity to cold (Raynaud's phenomenon), muscle weakness,muscle fatigue, changes in skin or tissue tone, shortness of breath orother abnormal breathing patterns, chest pain or constriction of thechest muscles, abnormal heart rate (e.g., elevated or lowered), lightsensitivity, blurry or otherwise abnormal vision, and reduced organfunction

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular inflammatory-relateddisorder. Alleviation of one or more symptoms of theinflammatory-related disorder indicates that the antibody confers aclinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

In another embodiment, antibodies directed against IL-6Rc and/or bothIL-6Rc and IL-6R may be used in methods known within the art relating tothe localization and/or quantitation of IL-6Rc (e.g., for use inmeasuring levels of IL-6Rc and/or both IL-6Rc and IL-6R withinappropriate physiological samples, for use in diagnostic methods, foruse in imaging the protein, and the like). In a given embodiment,antibodies specific to IL-6Rc and/or both IL-6Rc and IL-6R, orderivative, fragment, analog or homolog thereof, that contain theantibody derived antigen binding domain, are utilized aspharmacologically active compounds (referred to hereinafter as“Therapeutics”).

In another embodiment, an antibody specific for IL-6Rc can be used toisolate an IL-6R, IL-6Rc, and/or IL-6 polypeptide, by standardtechniques, such as immunoaffinity, chromatography orimmunoprecipitation. Antibodies directed against the IL-6Rc and/or bothIL-6Rc and IL-6R protein (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In yet another embodiment, an antibody according to the invention can beused as an agent for detecting the presence of IL-6Rc and/or both IL-6Rcand IL-6R protein (or a protein fragment thereof) in a sample. In someembodiments, the antibody contains a detectable label. Antibodies arepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof (e.g., F_(ab), scFv, or F_((ab)2)) is used. The term“labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently-labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently-labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Included within the usage of the term“biological sample”, therefore, is blood and a fraction or component ofblood including blood serum, blood plasma, or lymph. That is, thedetection method of the invention can be used to detect an analyte mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of an analyte mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of an analyte protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of an analytegenomic DNA include Southern hybridizations. Procedures for conductingimmunoassays are described, for example in “ELISA: Theory and Practice:Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) HumanPress, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and“Practice and Theory of Enzyme Immunoassays”, P. Tijssen, ElsevierScience Publishers, Amsterdam, 1985. Furthermore, in vivo techniques fordetection of an analyte protein include introducing into a subject alabeled anti-analyte protein antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

Therapeutic Administration and Formulations of huIL-6Rc Antibodies

The antibodies of the invention (also referred to herein as “activecompounds”), and derivatives, fragments, analogs and homologs thereof,can be incorporated into pharmaceutical compositions suitable foradministration. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington's Pharmaceutical Sciences: TheScience And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al.,editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement:Concepts, Possibilities, Limitations, And Trends, Harwood AcademicPublishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery(Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically comprise the antibody and a pharmaceuticallyacceptable carrier. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.(See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993)).

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration.Suitable carriers are described in the most recent edition ofRemington's Pharmaceutical Sciences, a standard reference text in thefield, which is incorporated herein by reference. Preferred examples ofsuch carriers or diluents include, but are not limited to, water,saline, ringer's solutions, dextrose solution, and 5% human serumalbumin. Liposomes and non-aqueous vehicles such as fixed oils may alsobe used. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe compositions is contemplated.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a sustained/controlled release formulations, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art.

For example, the active ingredients can be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

The materials can also be obtained commercially from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to infected cells with monoclonal antibodies to viralantigens) and can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the active compounds are administered in combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer, autoimmune disorders and inflammatorydiseases. The term “in combination” in this context means that theagents are given substantially contemporaneously, either simultaneouslyor sequentially. If given sequentially, at the onset of administrationof the second compound, the first of the two compounds is preferablystill detectable at effective concentrations at the site of treatment.

For example, the combination therapy can include one or more antibodiesof the invention coformulated with, and/or coadministered with, one ormore additional therapeutic agents, e.g., one or more cytokine andgrowth factor inhibitors, immunosuppressants, anti-inflammatory agents,metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostaticagents, as described in more detail below. Such combination therapiesmay advantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

Preferred therapeutic agents used in combination with an antibody of theinvention are those agents that interfere at different stages in aninflammatory response. In one embodiment, one or more antibodiesdescribed herein may be coformulated with, and/or coadministered with,one or more additional agents such as other cytokine or growth factorantagonists (e.g., soluble receptors, peptide inhibitors, smallmolecules, ligand fusions); or antibodies or antigen binding fragmentsthereof that bind to other targets (e.g., antibodies that bind to othercytokines or growth factors, their receptors, or other cell surfacemolecules); and anti-inflammatory cytokines or agonists thereof.

In other embodiments, the antibodies of the invention are used asvaccine adjuvants against autoimmune disorders, inflammatory diseases,etc. The combination of adjuvants for treatment of these types ofdisorders are suitable for use in combination with a wide variety ofantigens from targeted self-antigens, i.e., autoantigens, involved inautoimmunity, e.g., myelin basic protein; inflammatory self-antigens,e.g., amyloid peptide protein, or transplant antigens, e.g.,alloantigens. The antigen may comprise peptides or polypeptides derivedfrom proteins, as well as fragments of any of the following:saccharides, proteins, polynucleotides or oligonucleotides,autoantigens, amyloid peptide protein, transplant antigens, allergens,or other macromolecular components. In some instances, more than oneantigen is included in the antigenic composition.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto IL-6Rc, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, generation of peptidetherapeutics, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies—one with aspecificity to IL-6Rc and/or both IL-6Rc and IL-6R and another to asecond molecule that are conjugated together, (ii) a single antibodythat has one chain specific to IL-6Rc and/or both IL-6Rc and IL-6R and asecond chain specific to a second molecule, or (iii) a single chainantibody that has specificity to IL-6Rc and/or both IL-6Rc and IL-6R anda second molecule. Such bispecific antibodies are generated usingtechniques that are well known for example, in connection with (i) and(ii) See e.g., Fanger et al. Immunol Methods 4:72-81 (1994) and Wrightet al. Crit, Reviews in Immunol. 12125-168 (1992), and in connectionwith (iii) See e.g., Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52(1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing IL-6Rc.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to IL-6Rc and/or bothIL-6Rc and IL-6R and antibodies thereto, such as the antibodies of theinvention or screening of peptide libraries, therapeutic peptides can begenerated that are directed against IL-6Rc and/or both IL-6Rc and IL-6R.Design and screening of peptide therapeutics is discussed in connectionwith Houghten et al. Biotechniques 13:412-421 (1992), Houghten PNAS USA82:5131-5135 (1985), Pinalla et al. Biotechniques 13:901-905 (1992),Blake and Litzi-Davis BioConjugate Chem. 3:510-513 (1992). Immunotoxinsand radiolabeled molecules can also be prepared, and in a similarmanner, in connection with peptidic moieties as discussed above inconnection with antibodies. Assuming that the IL-6Rc and/or both IL-6Rcand IL-6R molecule (or a form, such as a splice variant or alternateform) is functionally active in a disease process, it will also bepossible to design gene and antisense therapeutics thereto throughconventional techniques. Such modalities can be utilized for modulatingthe function of IL-6Rc. In connection therewith the antibodies of thepresent invention facilitate design and use of functional assays relatedthereto. A design and strategy for antisense therapeutics is discussedin detail in International Patent Application No. WO 94/29444. Designand strategies for gene therapy are well known. However, in particular,the use of gene therapeutic techniques involving intrabodies could proveto be particularly advantageous. See e.g., Chen et al. Human GeneTherapy 5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997).General design of and considerations related to gene therapeutics isalso discussed in International Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the IL-6Rc molecule and itsinteractions with other molecules in accordance with the presentinvention, such as the antibodies of the invention, and others can beutilized to rationally design additional therapeutic modalities. In thisregard, rational drug design techniques such as X-ray crystallography,computer-aided (or assisted) molecular modeling (CAMM), quantitative orqualitative structure-activity relationship (QSAR), and similartechnologies can be utilized to focus drug discovery efforts. Rationaldesign allows prediction of protein or synthetic structures which caninteract with the molecule or specific forms thereof which can be usedto modify or modulate the activity of IL-6Rc. Such structures can besynthesized chemically or expressed in biological systems. This approachhas been reviewed in Capsey et al. Genetically Engineered HumanTherapeutic Drugs (Stockton Press, NY (1988)). Further, combinatoriallibraries can be designed and synthesized and used in screeningprograms, such as high throughput screening efforts.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with the binding of IL-6Rcto gp130, or candidate or test compounds or agents that modulate orotherwise interfere with the signaling function of the IL-6 receptor.Also provided are methods of identifying compounds useful to treatdisorders associated with aberrant IL-6 signaling. The invention alsoincludes compounds identified in the screening assays described herein.

In one embodiment, the invention provides assays for screening candidateor test compounds which modulate the signaling function of IL-6Rc. Thetest compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. (See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the signalingfunction of IL-6Rc and/or the interaction between IL-6 and IL-6R. Forexample, the antibody is monoclonal antibody 39B9 VL1 and the antigen isIL-6R. Alternatively, the monoclonal antibody is 39B9 VL5, 12A, or 5C,and the antigen is IL-6Rc or IL-6R.

In another embodiment, a soluble IL-6Rc and/or both IL-6Rc and IL-6Rprotein of the invention is provided and exposed to at least oneneutralizing monoclonal antibody. Formation of an antibody-antigencomplex is detected, and one or more candidate compounds are introducedto the complex. If the antibody-antigen complex is disrupted followingintroduction of the one or more candidate compounds, the candidatecompounds is useful to treat disorders associated with aberrant IL-6signaling.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a neutralizing antibody,such as monoclonal antibody 39B9 VL1, 39B9 VL5, 12A and 5C, each ofwhich modulates or otherwise interferes with IL-6 mediated activation ofthe JAK/STAT pathway and/or MAPK cascade.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use of either the soluble form or the membrane-bound form ofIL-6Rc and/or both IL-6Rc and IL-6R, and fragments thereof. In the caseof cell-free assays comprising the membrane-bound forms of IL-6Rc and/orboth IL-6Rc and IL-6R it may be desirable to utilize a solubilizingagent such that the membrane-bound form of the proteins are maintainedin solution. Examples of such solubilizing agents include non-ionicdetergents such as n-octylglucoside, n-dodecylglucoside,n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminio-1-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody (e.g. 39B9 VL1, 39B9 VL5, 12A and 5C) or the antigen (e.g.IL-6Rc and/or both IL-6Rc and IL-6R protein) can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated antibodyor antigen molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well-known within the art(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, other antibodies reactive with the antibody orantigen of interest, but which do not interfere with the formation ofthe antibody-antigen complex of interest, can be derivatized to thewells of the plate, and unbound antibody or antigen trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic and Prophylactic Formulations

The huIL-6Rc MAbs of the invention are used in diagnostic andprophylactic formulations. In one embodiment, an IL-6Rc and/or bothIL-6Rc and IL-6R antagonist, such as a huIL-6Rc MAb of the invention, isadministered to patients that are at risk of developing one or more ofthe aforementioned diseases, such as for example, without limitation,sepsis, cancer (e.g., multiple myeloma disease (MM), renal cellcarcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferativedisorder (BLPD), and prostate cancer), bone resorption, osteoporosis,cachexia, psoriasis, mesangial proliferative glomerulonephritis,Kaposi's sarcoma, AIDS-related lymphoma, and inflammatory diseases(e.g., rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus). A patient's or organ'spredisposition to one or more of the aforementioned autoimmune orinflammatory diseases can be determined using genotypic, serological orbiochemical markers.

In another embodiment of the invention, an IL-6Rc and/or both IL-6Rc andIL-6R antagonist, such as a huIL-6Rc antibody is administered to humanindividuals diagnosed with a clinical indication associated with one ormore of the aforementioned diseases, such as for example, withoutlimitation, sepsis, cancer (e.g., multiple myeloma disease (MM), renalcell carcinoma (RCC), plasma cell leukaemia, lymphoma,B-lymphoproliferative disorder (BLPD), and prostate cancer), boneresorption, osteoporosis, cachexia, psoriasis, mesangial proliferativeglomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, andinflammatory diseases (e.g., rheumatoid arthritis, systemic onsetjuvenile idiopathic arthritis, hypergammaglobulinemia, Crohn's disease,ulcerative colitis, systemic lupus erythematosus (SLE), multiplesclerosis, Castleman's disease, IgM gammopathy, cardiac myxoma, asthma,allergic asthma and autoimmune insulin-dependent diabetes mellitus).Upon diagnosis, an IL-6Rc and/or both IL-6Rc and IL-6R antagonist, suchas a huIL-6Rc antibody is administered to mitigate or reverse theeffects of the clinical indication associated with one or more of theaforementioned diseases, such as for example, without limitation,sepsis, cancer (e.g., multiple myeloma disease (MM), renal cellcarcinoma (RCC), plasma cell leukaemia, lymphoma, B-lymphoproliferativedisorder (BLPD), and prostate cancer), bone resorption, osteoporosis,cachexia, psoriasis, mesangial proliferative glomerulonephritis,Kaposi's sarcoma, AIDS-related lymphoma, and inflammatory diseases(e.g., rheumatoid arthritis, systemic onset juvenile idiopathicarthritis, hypergammaglobulinemia, Crohn's disease, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, asthma, allergic asthma andautoimmune insulin-dependent diabetes mellitus).

Antibodies of the invention are also useful in the detection of IL-6Rcand/or both IL-6Rc and IL-6R in patient samples and accordingly areuseful as diagnostics. For example, the huIL-6Rc antibodies of theinvention are used in in vitro assays, e.g., ELISA, to detect IL-6Rcand/or both IL-6Rc and IL-6R levels in a patient sample.

In one embodiment, a huIL-6Rc antibody of the invention is immobilizedon a solid support (e.g., the well(s) of a microtiter plate). Theimmobilized antibody serves as a capture antibody for any IL-6Rc and/orboth IL-6Rc and IL-6R that may be present in a test sample. Prior tocontacting the immobilized antibody with a patient sample, the solidsupport is rinsed and treated with a blocking agent such as milk proteinor albumin to prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of IL-6Rc and/or both IL-6Rc and IL-6R antigen in the testsample is determined by comparison with a standard curve developed fromthe standard samples.

It will be appreciated that based on the results obtained using thehuIL-6Rc antibodies of the invention in an in vitro diagnostic assay, itis possible to stage a disease (e.g., a clinical indication associatedwith ischemia, an autoimmune or inflammatory disorder) in a subjectbased on expression levels of the IL-6Rc and/or both IL-6Rc and IL-6Rantigen. For a given disease, samples of blood are taken from subjectsdiagnosed as being at various stages in the progression of the disease,and/or at various points in the therapeutic treatment of the disease.Using a population of samples that provides statistically significantresults for each stage of progression or therapy, a range ofconcentrations of the antigen that may be considered characteristic ofeach stage is designated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1: Cloning, Expression and Purification of Interleukin-6 (IL-6),IL-6 Receptor (IL-6R) and the Complex of IL-6/IL-6R (IL-6Rc)

The cDNAs encoding the human IL-6R (Accession No. X12830), human IL-6(Accession No. BC015511), cynomolgus IL-6R, cynomolgus IL-6 (AccessionNo. AB000554), mouse IL-6R (Accession No. NM_010559) and mouse IL-6(Accession No. NM_031168) were amplified by PCR from peripheral bloodmononuclear cells (PMBC)-derived cDNA and cloned in PCR4TOPO vector(Invitrogen). Following a subsequent PCR step, a His- or Avi-Tag(Avidity, Denver Colo.) was introduced at the C-terminus of the cytokinecoding sequence. These constructs were then sub-cloned intocorresponding vectors for expression of either the soluble or membraneforms of IL-6, IL-6R and IL-6Rc. The soluble human IL-6Rc (shuIL-6Rc)recombinant protein was generated by fusing IL-6 to IL-6R (the fusionprotein of IL-6/IL-6R taken from Mizuguchi et al., 2001 J. Biosci.Bioeng. 91(3):299-304 with modifications as follows: aa1-333 for IL-6Rand aa28-212 for IL-6). His-tagged coding sequences of cytokines fromvarious species (human, cynomolgus, mouse) were placed under the controlof the EF1 promoter and/or CMV promoter for expression in the episomalexpression vector pEAK8 or pEE14.4 for soluble forms. Thecytokine-coding sequence was followed by a viral internal ribosome entrysite (IRES) and a second or third cistron for the co-expression of BirAand GFP. The pEAK8 vector contains the puromycin resistance gene, theEBV nuclear antigen 1 (EBNA1) and the oriP origin of replication. EBNA1and oriP are necessary for the propagation of the pEAK8 vector asepisomal DNA in human cells and the generation of stable transfectants.Stably transfected cells were obtained after 7-10 days of culture in thepresence of 2 ug/mL of puromycin. Puromycin-resistant cells wereexpanded and used for soluble cytokine production. The biologicalactivity of the soluble His-tagged, human, mouse and cynomolgus, IL-6R,IL-6 and IL-6Rc was tested in various functional assays and found to becomparable to reagents from commercial sources (where available). Forcell surface expression the IL-6R, IL-6 and IL-6Rc constructs werecloned into the pDisplay vector, transected into CHO cells and selectedwith G418 selection.

Example 2: Immunizations

Fully human monoclonal antibodies were generated using transgenicstrains of mice in which mouse antibody gene expression was suppressedand replaced with human antibody gene expression. Three strains oftransgenic mice were used:

-   -   1) HuMab® mouse (Medarex, Princeton N.J.)    -   2) KM™ mouse, a crossbred between HuMAb Mouse and Kirin's TC        Mouse (Kirin Pharma Company, Japan)    -   3) KM (FCγRIIb-KO) mouse, a strain derived from KM™ mouse, in        which the gene Fcgr2b coding for the inhibitory Fc gamma        Receptor IIB has been inactivated.

Mice were immunized either with Chinese Hamster Ovary expressing humanIL-6Rc at the cell surface (CHO/IL-6Rc) or with soluble human IL-6Rc(shuIL-6Rc).

In general, all animals received from 7 to 10 injectionsintraperitoneally (i.p.) or subcutaneously (s.c.) with CHO/IL-6Rc orIL-6Rc emulsified in MPL+TDM adjuvant (RIBI) as described herein. Theinitial 5 to 8 injections were all done in the presence of adjuvant. Thetwo final hyperboosts preceding the fusion were done with free antigenand without adjuvant. An example of a representative immunizationschedule:

Day 1 10⁷ CHO/IL-6Rc cells, i.p. in RIBI Day 14 10⁷ CHO/IL-6Rc cells,i.p. in RIBI Day 28 10⁷ CHO/IL-6Rc cells, i.p. in RIBI Day 42 50 μgshuIL-6Rc, i.p. in RIBI Day 56 20 μg shuIL-6Rc, s.c. in RIBI Day 70 10μg shuIL-6Rc, s.c. in PBS Day 84 10 μg shuIL-6Rc, s.c. in PBS Day 87 5μg shuIL-6Rc, s.c. in PBS

Sera of immunized animals were screened periodically by flow cytometricanalysis to detect the presence of human IgG directed to CHO/IL-6Rc ascompared to CHO cells alone. To obtain hybridomas, popliteal, inguinal,para-aortic, submandibular, cervical, axial, and brachial lymph nodeswere removed from the mice and digested with collagenase and DNAse.Single cells suspension of lymph node cells was mixed at 1:1 ratio withSP2/0 myeloma cells and suspended in Cytofusion Low Conductivity Medium(CPS-LCMC, CytoPulse Sciences, Inc.). Fusions were done with 30 to 60million splenocytes in the CytoPulse CEEF50 Electrofusion apparatus asindicated by the manufacturer (Cyto Pulse Sciences, Inc.). Afterelectrofusion, cells were incubated for approximately 1 hour at 37° C.to allow recovery before distributing into 96-well plates. Fused cellswere resuspended in HAT selection medium and plated in 44 to 52 96-wellplates at a cell concentration of 0.1-0.2×10⁵ splenocytes per well in200 μl medium. Hybridoma selection proceeded for 14 days. Fusion oflymph nodes of immunized mice resulted in the generation of hybridomasproducing antibodies specific to IL-6Rc. Fourteen days after the fusion,hybridoma-containing plates were screened for the presence of human IgGbinding to human CHO/IL-6Rc.

Example 3: Biological Assays for IL-6Rc Activity

All assays described herein were performed in parallel with a controlhuman IgG₁ monoclonal antibody to human IL-6R (U.S. Pat. No. 5,817,790,SEQ ID NO:69 and SEQ ID NO: 71); hereto referred as “control mAb”.Furthermore, the 39B9 V_(L)1 antibody (nucleic acid sequences SEQ ID NO:1 and 3, amino acid sequences SEQ ID NO: 2 and 4) has been designatedthe nomenclature “NI-1201”.

On target cells, IL-6 first binds to the membrane-bound IL-6R (mIL-6R).The complex of IL-6/IL-6R associates with the signal-transducingmembrane protein gp130, thereby promoting its dimerization and thesubsequent initiation of intracellular signaling. gp130 is ubiquitouslyexpressed by cells whereas mIL-6R has a reduced expression profile onhepatocytes and a restricted number of immune cells. A naturallyoccurring, soluble form of the IL-6R (sIL6R) is generated by proteolysisof the membrane form or by differential splicing of IL-6R mRNA. ThesIL-6R combines with IL-6 to form the soluble IL-6/IL-6R complex(IL-6Rc) and is capable of activating mIL-6R-negative gp130-positivecells. This mechanism is called trans-signaling whereas signaling viaIL-6 binding to mIL-6R and subsequent coupling to gp130 is termedcis-signaling (Taga et al., 1989, Cell, 58: 573-581).

IL-6 Functional Assays:

BAF-hugp130 cells (BAF-130), a human gp130-transfected mouse pro-B cellline, proliferate in the presence of IL-6 and shuIL-6R (FIG. 1).Similarly, BAF-130 cells transfected with membrane bound huIL-6R(BAF-130/IL-6R) proliferate when cultured with huIL-6 (FIG. 2).

For trans-signaling analysis, the “native” IL-6Rc (as opposed torecombinant the fusion complex) was formed by incubating the cytokine(IL-6) with its cognate soluble receptor (shuIL-6R) at 37° C. for 3-4 h.Several concentrations of mAbs were added on cells before culturing withthe native complex. BAF-130 cells (1×10⁴ cells/0.2 mL/well) wereincubated for 72 h in a 96-well flat-bottom plate in RPMI supplementedwith 0.5% fetal calf serum in the presence of test mAbs (Control mAb,huIgG1 or NI-1201) with huIL-6+shuIL-6R (FIG. 1A-D). Proliferation wasassessed using the cell proliferation reagent WST-1 (Roche) according tomanufacturer's instructions. Briefly, after the culture period, 20 μL ofWST-1 reagent were added in medium and incubated at 37° C. 5% CO2 for 4hours. Absorbance (450-650 nm) was measured using a microplate reader.Results demonstrate that NI-1201 neutralizes the activity of the nativeIL-6Rc more effectively than the control mAb.

For cis-signaling analysis, BAF-130/IL-6R cells were incubated withdifferent doses of mAbs and a fixed amount of IL-6 (FIG. 2A).Conversely, cells were also incubated with one concentration of mAb inthe presence of ascending amounts of IL-6 (FIG. 2B). Proliferation wasassessed using the cell proliferation reagent WST-1 as above. NI-1201and control mAb demonstrate equivalent activity in blocking thiscis-signaling assay.

Example 4: Variants of huIL-6Rc Antibodies

Variants of the huIL-6Rc antibodies are made using any of a variety ofart-recognized techniques. For example, variant huIL-6Rc antibodiesinclude antibodies having one or more amino acid modifications, such as,for example, an amino acid substitution, at position within the antibodysequence.

Preferred locations for amino acid substitutions are shown as bold,underlined residues below in Table 3. The amino acid residues inbold/underline can be replaced with any amino acid residue. In preferredembodiments, the amino acid residues in bold/underline are replaced withthe amino acid residues shown below in Table 3. In these embodiments,the antibody comprises (i) the consensus amino acid sequence QQSXSYPLT(SEQ ID NO: 42) in the light chain complementarity determining region 3(CDR3), where X is N or Q; (ii) the consensus amino acid sequenceGIIPX₁FX₂TTKYAQX₃FQG (SEQ ID NO: 43) in the heavy chain complementaritydetermining region 2 (CDR2), where X₁ is L or A, X₂ is D or E, and X₃ isQ or K; (iii) the consensus amino acid sequence DRDILTDYYPXGGMDV (SEQ IDNO: 44) in the heavy chain complementarity determining region 3 (CDR3),where X is M or L; and (iv) the consensus amino acid sequence TAVXYCAR(SEQ ID NO: 45) in the framework region 3 (FRW3), where X is F or Y.

The NI-1201-wild type (NI-1201-WT) antibody listed in Table 3 comprisesthe amino acid sequence QQSNSYPLT (SEQ ID NO: 26) in the light chainCDR3 region, the amino acid sequence GIIPLFDTTKYAQQFQG (SEQ ID NO: 16)in the heavy chain CDR2 region, the amino acid sequence DRDILTDYYPMGGMDV(SEQ ID NO: 36) in the heavy chain CDR3 region, and the amino acidsequence TAVFYCAR (SEQ ID NO: 38) in the FRW3 region.

The NI-1201-A antibody listed in Table 3 comprises the amino acidsequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, theamino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 33) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPMGGMDV (SEQ ID NO: 36) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-B antibody listed in Table 3 comprises the amino acidsequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, theamino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 33) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-C antibody listed in Table 3 comprises the amino acidsequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, theamino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 34) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-D antibody listed in Table 3 comprises the amino acidsequence QQSQSYPLT (SEQ ID NO: 32) in the light chain CDR3 region, theamino acid sequence GIIPAFETTKYAQKFQG (SEQ ID NO: 34) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-E antibody listed in Table 3 comprises the amino acidsequence QQSQSYPLT (SEQ ID NO: 32) in the light chain CDR3 region, theamino acid sequence GIIPLFDTTKYAQKFQG (SEQ ID NO: 33) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-F antibody listed in Table 3 comprises the amino acidsequence QQSNSYPLT (SEQ ID NO: 26) in the light chain CDR3 region, theamino acid sequence GIIPAFDTTKYAQKFQG (SEQ ID NO: 35) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

The NI-1201-G antibody listed in Table 3 comprises the amino acidsequence QQSQSYPLT (SEQ ID NO: 32) in the light chain CDR3 region, theamino acid sequence GIIPAFDTTKYAQKFQG (SEQ ID NO: 35) in the heavy chainCDR2 region, the amino acid sequence DRDILTDYYPLGGMDV (SEQ ID NO: 37) inthe heavy chain CDR3 region, and the amino acid sequence TAVYYCAR (SEQID NO: 39) in the FRW3 region.

TABLE 3 NI-1201 Lead Candidates Light chain CDR3 Heavy chain CDR2Heavy chain CDR3 FRW 3 NI1201-WT QQSNSYPLT GIIPLFDTTKYAQQFQGDRDILTDYYPMGGMDV ...TAVFYCAR... NI1201-A QQSNSYPLT GIIPLFDTTKYAQ K FQGDRDILTDYYPMGGMDV ...TAV Y YCAR... NI1201-B QQSNSYPLT GIIPLFDTTKYAQ K FQGDRDILTDYYP L GGMDV ...TAV Y YCAR... NI1201-C QQSNSYPLT GIIP A F E TTKYAQK FQG DRDILTDYYP L GGMDV ...TAV Y YCAR... NI1201-D QQS Q SYPLT GIIP A FE TTKYAQ K FQG DRDILTDYYP L GGMDV ...TAV Y YCAR... NI1201-E QQS Q SYPLTGIIPLFDTTKYAQ K FQG DRDILTDYYP L GGMDV ...TAV Y YCAR... NI1201-FQQSNSYPLT GIIP A FDTTKYAQ K FQG DRDILTDYYP L GGMDV ...TAV Y YCAR...NI1201-G QQS Q SYPLT GIIP A FDTTKYAQ K FQG DRDILTDYYP L GGMDV ...TAV YYCAR...

Example 5: NI-1201 Blocks STAT-3 Phosphorylation Induced by IL-6Cis-Signaling

After a 24 h serum starvation, the mIL-6R-positive human hepatocellularcarcinoma HepG2 cells (ATCC) were incubated for 10 min with indicatedconcentrations of hIL-6 (FIG. 3A) or with 10 ng/mL IL-6 with indicatedconcentrations of huIgG1, control mAb or NI-1201 WT (FIG. 3B). Afterlysis in sample buffer, proteins were analyzed in SDS-PAGE westernblotting using a monoclonal anti-phospho-STAT3 antibody, P-STAT3 (Cellsignaling technology). The blots were stripped and re-probed with apolyclonal antibody recognizing activated and non-activated STAT3 (SantaCruz Biotechnology). NI-1201 and control mAb demonstrated equivalentactivity in blocking the cis-signaling induced phosphorylation ofSTAT-3.

Example 6: NI-1201 Blocks IL-6 Trans-Signaling Mediated by shuIL-6Rc

PEAK cells were transiently transfected with the Luciferase reporterplasmid (promoter STAT3 dependent). 2.5×10⁴ cells per well were seededinto 96-well flat-bottomed plates in DMEM containing 0.5% fetal calfserum. After 4-6 h cell adhesion, PEAK cells were activated with 30ng/mL of shuIL-6Rc with ascending doses of indicated mAbs. 18 h later,medium was removed and the Luciferase assay was carried out using thesteady-Glo® Luciferase Assay system (Promega) in a chemoluminescenceanalyzer. All variants of NI-1201 neutralized, in a dose dependentfashion, the activity of shuIL-6Rc whereas the control mAb failed toblock the activity of the pre-formed complex (FIG. 4).

Example 7: Affinity and Binding Kinetics of huIL-6Rc Antibodies

The ability of NI-1201 to bind to native membrane bound huIL-6R wasevaluated using flow cytometric analysis on the HepG2 hepatoma cellline. Cell surface staining is performed on HepG2 cells with differentdoses of mAbs. Binding of primary unconjugated mAbs (Control mAb, huIgG1and NI-1201 mAbs) was detected with goat anti-human Alexa Fluor 647 IgG(H+L) (Invitrogen). Each experiment was performed in triplicates. Themean of fluorescence intensity (MFI) is represented and demonstrates anapparent increased affinity of NI-1201 for the membrane IL-6R ascompared to the control mAb (FIG. 5).

The affinity and binding kinetics of NI-1201 candidates (A-D), NI-1201WT and control mAb were characterized on a Biacore 2000 instrument(Biacore AB, Uppsala, Sweden). A CMS Biacore chip was used and 1640 RU(response units) of an anti-human IgG Fc (Biacore AB, Uppsala, Sweden)was immobilized by EDC/NHS chemistry. This surface was used to captureNI-1201 candidates, NI-1201 WT and the control mAb. The surface wasregenerated after each cycle by injection of 3M magnesium chloride at 20μL/min, for 30 s followed by lmin of stabilization time in HBS-EP buffer(Biacore AB, Uppsala, Sweden). Binding was measured by passing analytescarrier-free soluble human IL-6R (shuIL6-R; R&D), soluble human IL-6Rc(shuIL-6Rc) and soluble cynomolgus monkey IL-6R (scyIL-6R) in duplicatesat the following concentrations: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nMand OnM. All solutions were diluted in HBS-EP buffer. Injection wasperformed at 50 μl/min for 3 min followed by 12 min of dissociation timeand the temperature was set at 25° C. The data were fitted according to1:1 Langmuir model and the K_(on), K_(off) and K_(D) values determined.The affinities and kinetic constants of NI-1201 variants A-D, NI-1201 WTand control mAb are summarized in the Table 4. Confirming the functionalassays using either native or preformed IL-6Rc, NI-1201 demonstrated asub-nanomolar affinity for the complex whereas the control mAb exhibitedno measurable binding.

TABLE 2 Kinetic and affinity constants measured by Biacore AnalyteSample K_(on) (1/Ms) K_(off) (1/s) K_(D) (M) shuIL-6R NI-1201 A 8.29E+051.10E−04 1.21E−10 NI-1201 B 8.75E+05 9.40E−05 1.07E−10 NI-1201 C9.92E+05 1.10E−04 1.10E−10 NI-1201 D 7.61E+05 1.02E−04 1.34E−10 NI-1201WT 1.33E+06 7.53E−05 5.67E−11 Control mAb 4.14E+05 3.99E−04 9.65E−10shuIL-6Rc NI-1201 A 5.01E+04 2.46E−04 4.92E−09 NI-1201 B 5.34E+042.37E−04 4.43E−09 NI-1201 C 4.77E+04 2.40E−04 5.03E−09 NI-1201 D4.53E+04 2.22E−04 4.89E−09 NI-1201 WT 4.31E+04 2.43E−04 5.64E−09 ControlmAb N.D. N.D. N.D. scyIL-6R NI-1201 A 1.04E+05 2.13E−04 2.05E−09 NI-1201B 1.15E+05 2.07E−04 1.81E−09 NI-1201 C 1.11E+05 2.25E−04 2.03E−09NI-1201 D 1.12E+05 2.00E−04 1.79E−09 NI-1201 WT 1.06E+05 2.15E−042.04E−09 Control mAb 2.68E+04 4.63E−04 1.73E−08 shuIL-6R = soluble humanIL-6 Receptor; shuIL-6Rc = soluble human IL-6/IL-6 Receptor complex;scyIL-6R = soluble cynomolgus monkey IL-6 Receptor; N.D. = no bindingdetected.

Example 8: Epitope Mapping of huIL-6Rc Antibodies

Construction of IL-6R Chimeras and Mutated Mouse IL-6R:

Each modification was performed on the soluble form of IL-6R and addedin a pDISPLAY™ vector (Invitrogen) allowing surface expression ofconstructs. Epitope mapping of NI-1201 was assessed by exchanging humanresidues in the second fibronectin II domain (D3 domain) of mouse IL-6Rand by verifying if binding of mAbs on modified mouse IL-6R wasrecovered.

An overlap extension PCR strategy was used to generate coding sequencesspecifying huIL-6R/mouseIL-6R chimeric proteins (FIG. 6A) and mouseIL-6R proteins containing human amino acid substitutions in D3 domains(FIG. 6B). Partially overlapping mutagenic oligonucleotide primers wereused for PCR amplification of N- and C-terminal sequences on either sideof huIL-6R/mouse IL-6R junctions or substituted regions, followed by gelisolation and annealing of the denatured PCR products. Full length PCRproducts were then digested by EcoRI and BglII and ligated into apDISPLAY™ vector (Invitrogen). PEAK cells were grown in DMEM mediumsupplemented with 10% fetal calf serum and 4 mM L-glutamine. Cells wereplated 12 to 24 h before transfection to have 40-50% confluency in 6well plates. Transfections of pDISPLAY™ vectors (Invitrogen) werecarried out by lipofection using TransIT® LT1 reagent (MirusBio)according manufacturer's specifications. Cells were grown for 48 hbefore harvesting and analysis by flow cytometry.

Facs Analysis:

Cell surface staining was performed on PEAK cells displaying eachvariant of IL-6R and analyzed by flow cytometry (FIG. 6B-D). Cellsurface expression of each chimeric IL-6R was assessed using ananti-human-CD126 PE or an anti-mouse CD126-PE antibody (BD Pharmingen).Control mAb and NI-1201 binding was detected using a goat anti-human IgG(H+L) Alexa Fluor 647 (Invitrogen). Data demonstrated that NI-1201recognizes a distinct epitope on huIL-6R to the Control mAb.

Example 9: NI-1201 Cross-Reacts and Neutralizes Cynomolgus Monkey IL-6Rc

IL-6R protein sequence homology between human and indicated species isshown (FIG. 7A). As described in example 6, PEAK cells were transientlytransfected with the Luciferase reporter plasmid (STAT3-dependentpromoter) and activated with 25 ng/mL cynomolgus IL-6+250 ng/mL scyIL-6Rwith different doses of indicated anti-human mAbs. Luciferase assay wascarried out as described above in Example 6 (FIG. 7B). Resultsdemonstrated the capacity of NI-1201 to neutralize the functionalactivity of native cynomolgus IL-6Rc.

What is claimed is:
 1. A method of treating an IL-6 signaling relateddisease, the method comprising administering to a subject an antibodythat binds to the IL-6/IL-6R complex (IL-6Rc), wherein the antibodycomprises a VH CDR1 region comprising the amino acid sequence of SEQ IDNO: 15, a VH CDR2 region comprising the amino acid sequence of SEQ IDNO: 33, a VH CDR3 region comprising the amino acid sequence of SEQ IDNO: 36, a VL CDR1 region comprising the amino acid sequence of SEQ IDNO: 24, a VL CDR2 region comprising the amino acid sequence of SEQ IDNO: 25, and a VL CDR3 region comprising the amino acid sequence of SEQID NO:
 26. 2. The method of claim 1, wherein the IL-6 signaling relateddisease is selected from an inflammation related disorder, sepsis, boneresorption, osteoporosis, cachexia, mesangial proliferativeglomerulonephritis, AIDS-related lymphoma, systemic onset juvenileidiopathic arthritis, hypergammaglobulinemia, ulcerative colitis,systemic lupus erythematosus (SLE), multiple sclerosis, Castleman'sdisease, IgM gammopathy, cardiac myxoma, and autoimmuneinsulin-dependent diabetes mellitus.
 3. The method of claim 2, whereinthe method alleviates one or more symptoms associated with aninflammation related disorder selected from fever, general malaise,fatigue, fever, pain, joint pain, tachypnea chills, cough, dyspnea, lunginfection, respiratory disease, headache, diarrhea, rash, anemia, orreduced organ function.